Serotonin neurons integrate GABA and dopamine inputs to regulate meal initiation

Abstract

Obesity is a growing global health epidemic with limited orally administered therapeutics. Serotonin (5-HT) is one neurotransmitter which remains an excellent target for new weight-loss therapies, but a gap remains in understanding the mechanisms involved in 5-HT produced in the dorsal Raphe nucleus (DRN) and its involvement in meal initiation. Using an optogenetic feeding paradigm, we showed that the 5-HT^DRN➔arcuate nucleus (ARH) circuit plays a role in meal initiation. Incorporating electrophysiology and ChannelRhodopsin-2-Assisted Circuit Mapping, we demonstrated that 5-HT^DRN neurons receive inhibitory input partially from GABAergic neurons in the DRN, and the 5-HT response can be enhanced by hunger. Additionally, deletion of the GABA_A receptor subunit in 5-HT neurons inhibits meal initiation with no effect on the satiation process. Finally, we identified the role of dopaminergic inputs via dopamine receptor D2 in enhancing the response to GABA-induced feeding. Thus, our results indicate that 5-HT^DRN neurons are inhibited by synergistic inhibitory actions of GABA and dopamine, for the initiation of a meal.

Keywords: Dopamine; GABA; Meal initiation; Serotonin.

Fig. 1:. The 5-HT^DRN→ARH circuit regulates meal initiation.

(A) Schematic of viral injection and optic fiber placement. (B) Schematic of blue light stimulation during food approaching. (C) Schematic of yellow light inhibition during food approaching. Results in fasted mice with blue light stimulation of the 5-HT^DRN→ARH circuit during food approaching (left): (D) food intake, (E) food bouts, (F) entries to the food approach zone, (G) probability of food intake during food approaching, (H) latency to eat, (I) bout duration. Results in sated mice with yellow light inhibition of the 5-HT^DRN→ARH circuit during food approach (right): (J) food intake, (K) food bouts, (L) entries to the food approach zone, (M) probability of food intake during food approaching, (N) latency to eat, (O) bout duration. Data were analyzed with a paired nonparametric Wilcoxon Test. Data are presented as mean ± SEM, n=11-13 male mice. p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.

Fig. 2:. GABA inhibition of 5-HT^DRN neurons is regulated by hunger.

(A) Schematic representation of viral approach. (B) Schematic of optogenetic electrophysiology recording. (C-E) Representative evoked inhibitory post-synaptic current (eIPSC) traces. (F) Quantification of eIPSC frequency from C-E. (G) Quantification of eIPSC amplitude from C-E. (H-J) Representative miniature inhibitory post-synaptic current (mIPSC) traces. (K-L) mIPSC amplitude quantification of 5-HT^DRN neurons from male (K) and female (L) mice in a fed, 24 h fasted and 24 h fasted followed by 2 h refeed (refed) state. Data were analyzed with a two-way ANOVA with post-hoc multiple comparisons. Data are presented as mean ± SEM. For F-G, n=9 cells from 4 male mice. For H-L, n=8-21 cells from 3-5 male and female mice. p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.

Fig. 3:. GABAergic actions in 5-HT^DRN neurons regulate meal initiation.

(A) Representative image of cannula placement above the DRN. (B) Cumulative food intake results in control mice after saline or Muscimol DRN infusion. (C) Cumulative food intake results in TPH2^DRNKO mice after saline or Muscimol DRN infusion. (D) Cumulative food intake results in control and TPH2^DRNKO mice after saline or Muscimol DRN infusion. (E) Average daily food intake, (F) meal interval, (G) meal number, and (H) meal size in γ2^TPH2KO mice and control littermates. Data were analyzed with a two-way ANOVA with post-hoc multiple comparisons, except (D) which was analyzed by two-sided unpaired t-test. Data are presented as mean ± SEM, (B) n=15 male mice, (C) n=8 male mice, (D-G) n=5-6 male mice. p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.

Fig. 4.. DRD2 enhances 5-HT^DRN neuronal responses to GABAergic inputs.

mIPSC amplitude in control, DRD2^TPH2KO, and DRD1^TPH2KO (A) fed males and (B) fed females. (C) 24 h fasted males and (D) females. (E) Total food intake in control and DRD2^TPH2KO male mice after saline or Muscimol DRN infusion. Control mIPSC amplitude is the same as presented in Fig. 2K-L. Control Muscimol response is the same as presented in Fig. 3B. Data were analyzed with a two-way ANOVA with post-hoc multiple comparisons. Data are presented as mean ± SEM, (A-D) n=8-21 cells from 3-5 male and female mice, (E) n=8-15 male mice. p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.